Method for genotyping microsatellite DNA markers

ABSTRACT

The present invention provides a high throughput method for genotyping microsatellite markers. The technology uses a combination of oligonucleotides in a selective ligation reaction that discriminates mono-, di-, tri-, tetra-, penta-, hexa-, hepta-, octa- and nona-nucleotide repeated alleles in amplified DNA from individuals.

FIELD OF THE INVENTION

The present invention relates to a method for genotyping microsatelliteDNA markers using ligation of at least three oligonucleotides.Specifically, the present invention provides a method for distinguishingallele content in mono-, di-, tri-, tetra-, penta-, hexa-, hepta-, octa-or nona-repeated DNA using combinations of at least threeoligonucleotides comprising a 5′ primer, a central primer and aplurality 3′ primers that hybridize to different alleles ofmicrosatellite DNAs.

BACKGROUND OF THE INVENTION

The analysis of variation among polymorphic DNA provides valuable toolsfor genetic studies in the development of genetic engineering, medicine,gene mapping and drugs. For example, variations in polymorphic DNAallows one to distinguish one individual of a population from another,or to assess the predisposition of an individual to a heritable diseaseor trait.

Two types of genetic markers widely used in genetic studies includemicrosatellites and single nucleotide polymorphisms (SNPs).Microsatellites are genomic regions that are distributed approximatelyevery 30 kilobases throughout the genome and that contain a variablenumber of tandemly repeated sequences of mono, di-, tri-, tetra-,penta-, hexa-, hepta-, octa- or nona-nucleotides. SNPs are foundapproximately every kilobase in the genome.

SNPs and microsatellites differ in primary DNA structure, relativegenome density and genetic information. For example, SNPs are moresuitable than microsatellites for genotyping with a high-density ofmarkers because of their distribution and the high sequence specificitypossessed by sequences adjacent to the SNP site. Yet, microsatellitesare more informative than SNPs because microsatellites typically possessfour to sixteen different alleles compared to only two alleles for SNPs.

Presently, the most commonly used methods for genotyping microsatellitemarkers are gel-based PCR fragment analysis (Shi et al., (1999) Mol.Diagn., 4: 343-351). These methods are relatively more labor-intensiveand time-consuming due to gel preparation and gel reading steps, thanthe method described in this invention. Moreover, the automated DNAgenotyping instruments are expensive compared to other forms ofdetection and do not address the gel reading problems resulting fromnucleotide compression. Other methods such as differential hybridizationare limited by hybridization to two or more microsatellite markers thatshare sequences. (see Korkko et al., 1998).

Oligonucleotide Ligation Assays (OLAs) have been used to detect SNPs(Baron et al., 1996, see also U.S. Pat. Nos. 5,242,794 and 5,866,337) ormutations in a gene (Landegren et al., 1988, U.S. Pat. Nos. 4,988,617and 6,025,139). These OLAs are designed to hybridize contiguously tosingle-stranded target DNA sequences. Recently, an OLA was developed togenotype microsatellites containing mono- and di-nucleotide repeats(Zirvi et al., 1999a, 1999b, U.S. Pat. No. 6,054,564, WO 98/03673,EP956359).

FIG. 1 demonstrates the methods employed in these previous OLA methods.For example, in the OLA method used for the detection of SNPs, twooligonucleotides are designed to hybridize to the region of the testedsite where ligation would occur. The principle in the OLA assay fordetection of mutations within a gene is to hybridize multiple shortoligonucleotides contiguously throughout the entire gene. An OLA methodfor genotyping mono- and dinucleotide repeats has been reported wherethe ligation of the two oligonucleotides was performed at the middle ofthe repeat. In all of these methods, the presence of a mismatch wouldprevent hybridization and ligation of the oligonucleotide at or near thelocation of the mismatch.

The major drawback of using OLA for genotyping microsatellites is thatligation is not a highly discriminating process and background noise canbe a significant problem. To circumvent this problem, modifiednucleotides (containing nucleoside analogs) near the ligation junctionare used to improve the stringency of both the hybridization and theligation. However, this raises the cost, because relatively long,specific oligonucleotides arc required for these assays. A method thatovercomes these disadvantages of OLA would make this approach simplerand more efficient and amenable to the comparative genotyping of pooledDNA samples.

SUMMARY OF THE INVENTION

The present invention relates to a method for genotyping differentmicrosatellite DNA markers using ligation of at least threeoligonucleotides. In previous inventions, the principle was to hybridizecombinations of two, long oligonucleotides contiguously in order tocover the whole sequence of the microsatellite. This requires the costlysynthesis of specific, long oligonucleotides for each microsatellite tobe genotyped. In addition, the use of modified nucleotides was necessaryto achieve specificity. The present invention has eliminated theseproblems by using combinations of at least three oligonucleotides foreach allele at a locus. Collections of limited numbers ofoligonucleotides can be used for genotyping many differentmicrosatellites, thus reducing the cost of oligonucleotide synthesis inlarge genotyping projects. In addition, by using combinations of threeoligonucleotides, a high degree of specificity is achieved without theneed to use modified nucleoside analogs.

The present invention comprises the steps of providing a samplecontaining microsatellite DNA; selecting combinations of at least threeoligonucleotides that comprise a 5′ primer, a central primer and a 3′primers; mixing the sample and primers such that the primers andmicrosatellite DNA hybridize; adding a ligating reagent; and detectingthe presence of ligation products that consist of combinations of threeoligonucleotides (5′ primer, central primer and 3′ primers) boundtogether as a single oligonucleotide with a contiguous sequence,reflecting the precise genotype of the microsatellite in the sample, andthus the allele(s) present. In particular, the 5′ primer comprises atleast 5 base pairs complementary to the 3′ flanking region of themicrosatellite target strand; the central primer is complementary to therepeated region of the microsatellite target strand DNA; and the 3′primers comprise sequences that are complementary to the 5′ flankingsequence of the microsatellite target strand with the addition, at the5′ end, varying numbers of repeat units. The nature and the number ofrepeat units comprising the central primer depend on the nucleotides inthe repeated sequence and the number of repeat units of the shortestallele at a given locus. Likewise, the number of repeat units added atthe 5′ end of the 3′ primer also depends on the nucleotides in therepeated sequence and the number and identity (length) of the alleles ina population.

The present invention further provides a method for genotyping a DNApool containing a mixture of different DNA samples of the samemicrosatellite wherein the microsatellite DNA includes mono-, di-, tri-,tetra- penta-, hexa-, hepta-, octa- or nona-nucleotide repeated alleles.The detection of the allele content of the microsatellite DNA markerwithin the pooled sample is determined by gel filtration,electrophoresis, mass spectrometry or a gel free analysis.

In one embodiment, the present invention provides a method forgenotyping different alleles of a microsatellite DNA wherein the 5′ endof the 5′ primer is labeled with a detectable label. In anotherembodiment, the method provides detection of different allele of amicrosatellite DNA wherein the 5′ or 3′ primer is covalently linked to afunctional group, wherein the functional group is capable ofspecifically binding to a component of a solid support.

The present invention will decrease cost and improve the experimentalquality needed to achieve genotyping using high density DNA pooling. Themethod uses the capacity of DNA ligase to join selectively designedadjacent oligonucleotides that hybridize to a given DNA template. Thecombination of specifically designed oligonucleotides for each allelewithin a marker will allow discrimination between the differentgenotypes.

DESCRIPTION OF THE FIGURES

FIG. 1: Prior Art Oligonucleotide Ligation Assay (OLA) Techniques

FIG. 1 shows other prior techniques for OLA. In particular, the topfigure demonstrates an assay for the detection of mutations within agene. (Landergren et al. 1988; U.S. Pat. Nos. 4,988,617 and 6,025,319).The middle figure demonstrates an OLA for the detection of SNPs. (Baronet al.; U.S. Pat. Nos. 5,242794 and 5,866,337). The bottom figuredemonstrates an OLA for genotyping mono- and dinucleotide repeats.(Zirvi et al., 1999a, 1999b, U.S. Pat. No. 6,054,564). The template is asingle-stranded DNA containing the complementary sequence which theoligonucleotides are hybridizing. The letter “L” points the ligationsites.

FIG. 2: Oligonucleotide Ligation Assay Principle

FIG. 2 describes the components of the oligonucleotides of the presentinvention and demonstrates the ligation method.

FIG. 3: Protocol of Present Invention

FIG. 3 shows an embodiment of the method of the present inventiondiagrammatically.

FIG. 4: Design of a Gel Free Assay

FIG. 4 shows a second embodiment of the present invention as a gel freedetection assay. In this scheme, the different oligonucleotidecombinations will give different signals thus indicating the allelecontent of the sample. P5 and ligated P5-PC will not provide any signal.

FIGS. 5A & B: Case 1: Genotyping a [CA]₁₃ Repeat

FIGS. 5A & B shows an example of the present invention for genotyping a[CA]₁₃ Repeat DNA.

FIGS. 6A & B: Case 2: Genotyping a [CA]₁₄ Repeat

FIGS. 6A & B shows an example of the present invention for genotyping a[CA]₁₄ repeat DNA.

FIG. 7: Results of Genotyping [CA]₁₃ and [CA]₁₄ Repeats

The top figure demonstrates the use of the present invention ingenotyping CA- repeated microsatellite; a [CA]₁₃ homozygous (left); a[CA]₁₄ homozygous (middle) and a [CA]₁₃₋₁₄ heterozygous, (right),respectively. The bottom figure shows three control lanes to demonstratethe specificity of the generated ligation products: the reaction mixturecontains all reagents except the P3 oligonucleotide (left lane), noligase (middle lane) and no DNA template (right lane).

DEFINITIONS

Throughout the description of the present invention, several terms areused that are specific to the technology of this field. For the sake ofclarity and to avoid any misunderstandings, these definitions areprovided:

Allele: At a given locus, a particular form of a gene or genotype,specifying one of all the possible forms of the character encoded bythis locus. A diploid genome contains two alleles at any given locus.

Discriminating Primers: A set of primers that are individually specificfor one allele, depending on the number of repeat units that the primerspossess at either their 3′ or 5′ end. These primers differentiatebetween the different alleles.

Functional group: A moiety of chemical or proteinaeous nature that isattached to either the 5′ end or the 3′ end of an oligonucleotideallowing the latter to be purified by affinity.

Genotype: Set of alleles at a specified locus.

Hybridization: The process by which two nucleic acids are linkedtogether by base pairing, forming a duplex DNA of complementarysequences. In this description, the hybridization occurs betweenoligonucleotides and the PCR-amplified template. The conditions of theassay are designed to reach a perfectly matched duplex between theoligonucleotides and the template.

Label: An attachment linked to an oligonucleotide that permits itsspecific detection via the signal emitted by the attachment.

Ligase: An enzyme that catalyzes the formation of a phosphodiester bondat the site of single-stranded break within a DNA duplex.

Ligation: The process catalyzed by a DNA ligase.

Locus: A specified region of the genome.

Microsatellite: DNA of eukaryotic cells comprising highly repetitive DNAsequences flanked by sequences unique to that locus. In thisdescription, microsatellite refers to mono-, di-, tri-, tetra penta-,hexa-, hepta-, octa-, or nona-nucleotide repeated regions.

Oligonucleotide: A short single-stranded deoxyribonucleic acid molecule.In this description, the. length of the oligonucleotides varies from 5bases to more than 32 bases.

Polymerase Chain Reaction (PCR) amplification: An enzymatic processresulting in the exponential amplification of specific region of a DNAtemplate. The process uses a thermostable polymerase, capable ofreplicating a DNA template from a primer. In the presence of twoprimers, the region between them is amplified following this process.

Thermostable ligase: A heat resistant enzyme capable of performingligase functions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for genotyping differentalleles of microsatellite DNA by using a combination of at least threeoligonucleotides for each allele on the locus. In particular, thepresent method comprises providing a sample containing themicrosatellite DNA; selecting combinations of at least threeoligonucleotides comprising a 5′ primer (P5), wherein the 5′ primercomprises at least 5 base pairs complementary to the 3′ flanking regionof the repeated region of the microsatellite DNA; a central primer (PC),wherein the central primer is complementary to the repeated region ofthe microsatellite DNA; and at plurality of 3′ primers([repeat-unit]_(n)P3), which comprises a sequence that is complementaryto the 5′ flanking sequence of the repeat region of the microsatellite,and which possesses at its 5′ end, a number (n=0, 1, 2 . . . ) of repeatunits of the microsatellite to genotype, in particular,cytosine-adenine, (hereinafter, “[CA]_(n)”) for a CA repeated DNA,mixing the sample and primers such that the primers and microsatelliteDNA hybridize; adding a ligating reagent; and detecting the presence offull ligation products that consist of the three oligonucleotide primerslinked together in a contiguous sequence. (see FIGS. 3-7).

The principle described in the present invention holds for any changesin the orientation, length and modifications of the oligonucleondes. Oneskilled in the art could easily design a scheme in which thediscriminating primers were the 5′ primers, P5[repeat-unit]_(n). Inparticular, the present invention further provides a plurality of 5′primers ([repeat-unit]_(u)P5) which comprise a sequence that iscomplementary to the 3′ flanking sequence of the repeat region of themicrosatellite, and which possesses at its 3′ end, a number (n=0, 1, 2 .. . ) of repent units of the microsatellite to genotype, in particular,cytosine-adenine, (hereinafter, “[CA]_(n)”) for a CA repeated DNA; acentral primer, PC, wherein the central primer is complementary to therepeated region of the target strand of the microsatellite DNA and and a3′ primer P3, of a fixed length, wherein P3 comprises at least 5 basepairs that are complementary to the 5′ flanking region of the repeatedregion of the target microsatellite DNA strand.

The design of the oligonucleotides for the present invention provides alow cost technology. In particular, the present invention providesoligonucleotides wherein the 5′ primer, P5, is at least a 5-mer whereina collection of 1024 oligonucleotides (N⁵=A, C, G, or T) cover allpossible sequences of this size. The 3′ primer can be a 5-mer orgreater, preferably a 10 to 17-mer (excluding the repeat units at its 5′end). The central primer or repeat oligonucleotides can be used for thegenotyping of many microsatellites of the same repeat type, and arelatively small collection of approximately 500 differentoligonucleotides are sufficient for genotyping of the vast majority ofmicrosatellite loci.

The present invention will not only increase the throughput of theprocess at low cost, but it will also increase the precision andaccuracy of genotyping, that is expected to allow cost-effectivegenotyping of pooled DNA samples. The present method uses the capacityof the DNA ligase to join selectively designed adjacent oligonucleotidesthat hybridize to a given DNA template. (see FIG. 3). Combination ofspecifically designed oligonucleotides for each allele in the marker inthe assay will allows determination of the allele content within the DNAsample.

Therefore, in one embodiment of the present invention, the ligationreagent is T4 DNA ligase or a thermostable ligase. However, one skilledin the art could use any ligase known in the field to facilitate theligation step.

The present invention uses combinations of three oligonucleotides, P5,PC and P3, wherein the 5′ primer, P5, hybridizes to the complementary 3′flanking region of the microsatellite, the central primer, PC,hybridizes to the complementary repeated region of the microsatelliteand the 3′ primers, [repeat-unit]_(n)P3, hybridize to the 5′complementary flanking region of the microsatellite. (see FIG. 2).

In a preferred embodiment, P5 comprises a 5-mer sequence complementaryto the flanking region of the repeat and is radio labeled at its 5′-end.Therefore, in one embodiment, the detecting step further compriseslabeling the 5′ end of the 5′ primer with a detectable label. PCcomprises the complementary sequence of the repeat and its length isdictated by the shortest allele found at that particular microsatellitemarker in the population. The [repeat-unit]_(n)P3 is an ensemble ofprimers with a core sequence complementary to the 5′ flanking sequenceof the target strand and with a varying number (n=0, 1, 2, 3, . . . ) ofrepeat units at its 5′ end. The number of repeat added at the 5′ end of[repeat-unit]_(n)P3 is for the genotyping of all the existing alleles ofthe microsatellite marker.

The use of the present invention to perform genotyping with pooled DNAwill increase the value of the results of the assay. Therefore, in oneembodiment of the present invention, the sample is a mixture ofdifferent samples containing a given microsatellite locus from a numberof individuals.

By way of example, the present invention demonstrates the genotyping ofthe D6S471 locus which is a dinucleotide [CA]-repeat microsatellitemarker harbouring four different alleles, [CA]₁₃, [CA]₁₄, [CA]₁₆ and[CA]₁₇. Dinucleotide markers represent a degree of complexity, intrinsicto their primary structure, suitable for the experimental developmentneeded to achieve both specificity and stability of DNA hybridization.The methodology developed in genotyping this locus is applicable togenotyping mono-, di-, tri-, tetra-, penta, hexa-, hepta-, octa- andnona-nucleotide microsatellites. Therefore, in another embodiment, thepresent invention, provides a method for genotyping differentmicrosatellite DNA at a locus by using a combination of at least threeoligonucleotides for each allele on the loci wherein the sample ofmicrosatellite DNA consist of mono-, di-, tri-, tetra-, penta, hexa-,hepta-, octa- and nona-repeated alleles.

In yet another embodiment, the sample is an amplified PCR fragment ofthe microsatellite DNA.

Other systems use only two oligonucleotides with one ligation eventwhich implies that for each microsatellite, there is a need tosynthesize long, specific oligonucleotide. In addition, modifiednucleosides are often used to improve stringency of the hybridizationstep. These conditions increase the cost of large scale genotypingprojects. The present invention uses combinations of threeoligonucleotides, P5, PC and [repeat-unit]_(n) P3 and two ligation tolink the three oligonucleotides together which improves the specificityand eliminates the need for long oligonucleotides. In addition,specificity is achieved without using modified nucleosides.

Following hybridization of the three oligonucleotides onto the template,the DNA ligase joins them. After passing the ligation products through aG-50 column, they are separated by gel electrophoresis. Since P5 isradiolabelled at its 5′ end (*), only two ligation products will bevisible upon exposure on a X-ray film, P5-PC and P5-PC-P3. While theformer is genotype-independent, the latter will be formed exclusivelywhen the particular combination of oligonucleotides hybridize perfectlyto a given template, thus reflecting the genotype or allele content ofthe locus. Therefore, in one embodiment of the present invention, thedetecting step comprises separating the ligation products according totheir size by gel electrophoresis.

However, a gel-free analysis system would provide automation andefficiency. In another embodiment, the present invention provides amethod for detecting the ligation products by a gel free system whereinthe gel free system comprises covalently linking a functional group tothe 5′ end of the 5′-primer, P5, wherein the functional group exhibitsspecific binding to a component of a solid support; and labeling the 3′end of the 3′-primers, [repeat-unit]_(n)P3, with different detectablelabels wherein the different labels yield different signals. (see FIG.4). The different 3′-labels for each 3′-primer will differentiate and/ordiscriminate between genotypes by revealing a different signal (orsignals) depending upon which oligonucleotide combination(s) will ligatetogether. In one embodiment, the detectable labels are fluorescentlabels. In still another embodiment, the functional group is biotin andthe solid support is coated with streptavidin.

In another embodiment, the present invention provides a method fordetecting the ligation products by a gel free system wherein the gelfree system comprises covalently linking a functional group to the 3′end of the 3′-primer, P3, wherein the functional group exhibits specificbinding to a component of a solid support; and labeling the 5′ end ofthe 5′-primers, [repeat-unit]_(n)P5 with different detectable labelswherein the different labels yield different signals. The different5′-labels for each 5′-primer will differentiate and/or discriminatebetween genotypes by revealing a different signal (or signals) dependingupon which oligonucleotide combination(s) will ligate together.Therefore, the different labels attached to the discriminating primerswould be attached to the 5′ end of P5[repeat-unit]_(n). In oneembodiment, the detectable labels are fluorescent labels. In stillanother embodiment, the functional group is biotin and the solid supportis coated with streptavidin.

The present invention further provides a kit for detection of thepresence of different alleles at a locus by using a combination of atleast three oligonucleotides wherein the oligonucleotides comprisesequences which are complementary to and hybridize with one strand ofthe repeat region of a microsatellite DNA and having terminal groupssuch that the oligonucleotides are ligatable to each other whenhybridized perfectly to the microsatellite DNA.

In one embodiment of the present invention, a kit for the detection ofthe presence of different microsatellite DNA at a locus comprisescombinations of at least three oligonucleotides and instructions for useof the oligonucleotides in a suitable container means, wherein theoligonucleotides comprise sequences which are complementary to andhybridize with one strand of the repeat region of a microsatellite DNA,wherein the oligonucleotides comprise terminal groups such that theoligonucleotides are ligatable to each other when hybridized to themicrosatellite DNA. In another embodiment, the kit comprises a 5′primer; a central primer; a plurality of 3′ primers, and a ligatingreagent. In another embodiment, at least of one of the oligonucleotidesis labeled with a detectable label. In a preferred embodiment, the labelis a fluorescent label.

In yet another embodiment, the present invention provides a kit fordetection of the presence of different alleles at a locus by separatingthe ligation products by a gel free system wherein the kit comprisescombinations of at least three oligonucleotides and instructions for useof the oligonucleotides in a suitable container means, wherein theoligonucleotides comprise at least one primer that is covalently-linkedto a functional group, wherein the functional group exhibits specificbinding to a component of a solid support and at least one other primeris labeled with different detectable labels, wherein the differentlabels yield different signals.

In another embodiment, the kit comprises a 5′ primer that is covalentlylinked to a functional group at its 5′ end of the 5′ primer; a centralprimer; a plurality of 3′ primers that are labeled at their 3′ end withdifferent detectable labels wherein the different labels yield differentsignals (discriminating P3-primers); a ligating reagent, and a solidsupport. In a preferred embodiment, the detectable labels are differentfluorescent labels. Still further, in another embodiment of the presentkit, the functional group is biotin and the solid support is coated withstreptavidin.

In another embodiment, the kit comprises a 3′ primer that is covalentlylinked to a functional group at its 3′ end of the 3′ primer; a centralprimer; a plurality of 5′ primers that are labeled at their 5′ end withdifferent detectable labels wherein the different labels yield differentsignals (discriminating P5-primers); a ligating reagent, and a solidsupport. In a preferred embodiment, the detectable labels are differentfluorescent labels. Still further, in another embodiment of the presentkit, the functional group is biotin and the solid support is coated withstreptavidin.

EXAMPLES

These examples demonstrate the genotyping of homozygous and heterozygoustemplates of 13 and 14 [CA] repeats. The oligonucleotides werephosphorylated where the 5′ primer, P5, was radiolabeled. The targettemplate was an enriched single stranded DNA resulting from anasymmetric PCR reaction of the microsatellite to genotype and waspurified on a Sephadex column (G-50 micro spin, Pharmacia). The DNAtemplate and the oligonucleotides were first heat-denatured (bytransferring the respective tubes in boiling water for three minute)s.The following components were mixed at room temperature: five picomolesof each of the oligonucleotides, 3 microliters of formamide (finalconcentration of 30%), 2 microliters of 5× ligase buffer (Gibco/BRL) and0.4 micrograms of SSBP (Single Stranded Binding Protein, Promega). Then,0.5 picomoles of the template was added to the mixture and 1 microlitersof ligase (8 units, Gibco/BRL) was added immediately after. After anincubation of 5 minutes at room temperature, the reaction was stopped byheating (three minutes at 95° C.). The ligation products were thenseparated by gel electrophoresis in a 12% denaturing polyacrylamide gel.Using these conditions, templates of [CA]₁₃ versus [CA]₁₄ and viceversa, as well as heterozygous templates of 13 and 14 CA (see FIG. 7)were specifically genotyped. No modified nucleosides were used in theseassays.

When P5, PC and P3 (without any [CA]₀ unit at its 5′ end) werehybridized onto a [CA]₁₃ template, there were no gaps or bulges due tounpairing bases and both junctions (P5-PC and PC-P3) are ligated. Thefull discriminatory product was generated. In contrast, when P5, PC and[CA]₁P3 are hybridized on a [CA]₁₃ template, the [CA] unit of [CA]₁P3bulged out, thus preventing ligation. Note that the bulged [CA] was alsofound at the P5-PC junction, but the ligated PC-[CA]₁P3 was not visibleupon exposure on an X-ray film.

Example I Genotyping [CA]₁₃

For example, when genotyping. a template of [CA]₁₃, only the combinationP5-PC-[CA]₀P3 ligate (see FIG. 5A) The combination P5-PC-[CA]₁P3 did notligate on this teraplate because a [CA] unit bulged out at the junctionof either P5-PC or PC-[CA]₁P3 thus preventing ligation event to occur(see FIG. 5B)

Example II Genotyping [CA]₁₄

When genotyping a template of [CA]₁₄, the ligated combination wasP5-PC-[CA]₁P3 see FIG. 6A ). The combination P5-PC-P3 did not ligate ana template of [CA]₁₄ because a gap of dinucleotide [CA] existed betweenP5 and PC or between PC and P3 (see FIG. 6B).

When P5-PC and [CA]₀P3 are hybridized onto a [CA]₁₄ template, there wasa gap of one [CA] unit at either the P5-PC or PC-[CA]₀P3 junction andthe full, discriminatory product was not generated. However, P5, PC and[CA]₁P3 hybridize perfectly on the [CA]₁₄template and the full productwas generated.

Example III Genotyping the D6S471 locus

The genotype of the D6S471 locus was determined as an example of thepresent invention. This locus was a dinucleotide [CA]-repeatmicrosatellite marker harbouring four different alleles, [CA]₁₃, [CA]₁₄,[CA]₁₆ and [CA]₁₇. Dinucleotide markers represent a degree ofcomplexity, intrinsic to their primary structure, suitable for theexperimental tuning needed to achieve both specificity and stability ofDNA hybridization. The methodology developed in genotyping this locus isthus applicable to mono-, di-, tri-, tetra, penta-, hexa-, hepta-, octa-and nona-nucleotide microsatellites.

In the case of D6S471, PC consists of [CA]₁₃. The various P3oligonucleotides were of different lengths and consist of a coresequence complementary to the 5′-repeat-adjacent sequence, and of anumber of n of [CA] units at its 5′ end (where n represents all numberof dinucleotide necessary to fill the sequence gap to PC, reflectingthus all alleles in the population at that locus).

In the present model case, there were four different P3 reflecting thefour alleles at the D6S471 locus. For example, when genotyping atemplate of [CA]₁₃, only the combination P5-PC-P3 ligated (see FIG. 5A).The combination P5-PC-[CA]₁P3 did not ligate on this template because a[CA] unit bulged out at the junction of either P5-PC or PC-[CA]₁P3 (seFIG. 5B). When genotyping a template of [CA]₁₄, The ligated combinationwas P5-PC-[CA]₁ P3 (see FIG. 6A.) The combination P5-PC-P3 did notligate on a template of [CA]₁₄ because a gap of diniucleotide [CA]existed between P5 and PC or between PC and [CA]₀P3 (see FIG. 6B).

This application incorporates by reference the following publications:

References

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WO 91/17239

WO 95/27078

WO 98/03673

EP 956359

What is claimed is:
 1. A method for genotyping different alleles of amicrosatellite DNA locus said microsatellite DNA locus having a repeatregion and further having a predetermined number of different alleles,each allele having a different number of microsatellite repeat units insaid repeat region, said method comprising: (a) providing a samplecontaining the microsatellite DNA locus; (b) providing a set ofoligonucleotides comprising: (i) a 5′ primer complementary to a 3′flanking region of the repeat region of the microsatellite DNA locus;(ii) a central primer comprising a number of primer repeat unitscomplementary to the repeat region corresponding to the allele havingthe least number of said microsatellite repeat units; and, (iii) anensemble of discriminating 3′ primers comprising a plurality of 3′primers each of which has the formula of XY_(n), wherein X is a sequencethat is complementary to the a 5′ flanking sequence of the repeat regionof the microsatellite DNA Y is a primer repeat unit located at the 5′end of each of said 3′ primers which contains said primer repeat unit,the primer repect unit complementary to the microsatellite repeat unit;and, n is the number of primer repeat units in any one of saiddiscriminating 3′ primers wherein n is a varying number selected suchthat a combination of oligonucleotides comprising said 5′ primer, saidcentral primer and any one of said discriminating 3′ primers iscomplementary to one of said predetermined number of different allelesof the DNA microsatellite locus; (c) mixing the sample and primer suchthat the primers and microsatellite DNA hybridize; (d) adding a ligatingreagent; (e) detecting the presence of a ligation product thatincorporates three oligonucleotides, said three oligonucleotidescomprising said 5′ primer, said central primer and one of saiddiscriminating 3′ primers; and (f) deducing the genotype of the sampleaccording to the identities of the oligonucleotides incorporated in aligation product.
 2. The method according to claim 1, wherein the sampleis a mixture of different samples containing different alleles of agiven microsatellite DNA locus.
 3. The method according to claim 1,wherein the microsatellite DNA locus comprises mono-, di-, tri-, tetra-,penta-, hexa-, hepta-, octa- and nona-nucleotide repeated alleles. 4.The method according to claim 1, wherein the sample is an amplified PCRfragment of the microsatellite DNA locus.
 5. The method according toclaim 1, wherein the oligonucleotides comprise of modified nucleosides.6. The method according to claim 1, wherein the selecting step furthercomprises labeling the 5′ end of the 5′ primer with a detectable label.7. The method according to claim 1, wherein the detecting step comprisesseparating the ligation products according to their size by gelelectrophoresis.
 8. The method according to claim 1, wherein theligation reagent is T4 DNA ligase or a thermostable ligase.
 9. Themethod according to claim 1, wherein the detecting step furthercomprises separating the ligation products by a gel free system whereinthe gel free system comprises covalently linking a functional group tothe 5′ end of the 5′ primer wherein the functional group exhibitsspecific binding to a component of a solid support; and labeling the 3′end of the 3′ primers with different detectable labels wherein thedifferent labels yield different signals.
 10. The method according toclaim 9, wherein the detectable labels are fluorescent labels.
 11. Themethod according to claim 9, wherein the functional group is biotin andthe solid support is coated with streptavidin.
 12. The method accordingto claim 1 wherein said 5′ primer is a single primer comprising a 5 basesequence and said central primer is a single primer.
 13. The methodaccording to claim 1 wherein the total number of discriminating 3′primers is equal to the number of different alleles.
 14. A method forgenotyping, different alleles of a microsatellite DNA locus, the locuscomprising a repeat region with a flanking region located at each end ofthe repeat region, the microsatellite DNA locus having a repeat regionand further having a predetermined number of different alleles, eachallele having a different number of microsatellite repeat units in saidrepeat region, said method comprising: (a) providing a sample confiningthe microsatellite DNA locus; (b) providing a set of oligonucleotidescomprising: (i) a single primer complementary to a flanking regionlocated at one end of the repeat region of the microsatellite DNA locus;(ii) a central primer complementary to the repeat region correspondingto the allele having the least number of sad microsatellite repeatunits; and, (iii) an ensemble of discriminating primers comprising aplurality of primers, each of which has the formula of XY_(n), wherein Xis a sequence that is complements to the flanking region located at theopposite end of the repeat region recited in step (b)(i); Y is a primerrepeat unit complementary to the microsatellite repeat unit; and n isthe number of primer repeat units in any one of said discriminatingprimers, wherein n is a varying number selected such that a combinationof oligonucleotides comprising said single primer, said central primerand any one of said discriminating primers is complementary to one ofsaid predetermined number of different alleles; (c) mixing the sampleand primers such that the primers and microsatellite DNA hybridize; (d)adding a ligating reagent; and (e) detecting the presence of a ligationproduct that incorporates three oligonucleotides, said threeoligonucleotides comprising said single primer, said central primer andone of said discriminating primers; (f) deducing the genotype of thesample according to the identities of the oligonucleotides incorporatedin a ligation product.
 15. The method according to claim 14 wherein saidsingle primer is a single primer comprising a 5 base sequence and saidcentral primer is a single primer.
 16. The method according to claim 14wherein the plurality of discriminating primers is equal in number tothe number of different alleles at the locus.
 17. The method accordingto claim 14 wherein the sample is a mixture of different samplescontaining different alleles of a given microsatellite DNA.
 18. Themethod according to clam 14 wherein the microsatellite DNA locuscomprises of mono-, di-, ti-, tetra-, penta-, hexa-, octa- and nonanucleotide repeat alleles.
 19. The method according to claim 14 whereinthe sample is an amplified DNA fragment of the microsatellite DNA locus.20. The method according to claim 14 wherein the oligonucleotidescomprise of modified nucleosides.
 21. The method according to claim 14wherein the step of providing a set of oligonucleotides furthercomprises labelling an end of the single primer.
 22. The methodaccording to claim 14 wherein the detecting step comprises separatingthe ligation product according to their size by gel electrophoresis. 23.The method according to claim 14 wherein the ligation reagent is T4 DNAligase or a thermostable ligase.
 24. The method according to claim 14wherein the detecting step further comprises separating the ligationproduct by a gel tree system wherein the gel free system comprisescovalently linking a functional group to the end of the single primerwherein the functional group exhibits specific binding to a component ofa solid support; and labelling an end of X with different detectablelabels wherein the different labels yield different signals.
 25. Themethod according to claim 24 wherein the detectable labels arefluorescent labels.
 26. The method according to claim 24 wherein thefunctional group is biotin and the solid support is coated withstreptavidin.